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J Biomed Biotechnol. 2006; 2006: 83672. Published online 2006 February 5. doi: 10.1155/JBB/2006/83672. | PMCID: PMC1559935 |
L1 Retrotransposons in Human Cancers Wolfgang A. Schulz* Department of Urology, Heinrich Heine University, Mooreustrasse 5, 40225 Düsseldorf, Germany Received August 31, 2005; Accepted October 16, 2005. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited. Retrotransposons like L1 are silenced in somatic cells by a
variety of mechanisms acting at different levels. Protective
mechanisms include DNA methylation and packaging into inactive
chromatin to suppress transcription and prevent recombination,
potentially supported by cytidine deaminase editing of RNA.
Furthermore, DNA strand breaks arising during attempted
retrotranspositions ought to activate cellular checkpoints, and L1
activation outside immunoprivileged sites may elicit immune
responses. A number of observations indicate that L1 sequences
nevertheless become reactivated in human cancer. Prominently,
methylation of L1 sequences is diminished in many cancer types and
full-length L1 RNAs become detectable, although strong expression
is restricted to germ cell cancers. L1 elements have been found to
be enriched at sites of illegitimate recombination in many
cancers. In theory, lack of L1 repression in cancer might cause
transcriptional deregulation, insertional mutations, DNA breaks,
and an increased frequency of recombinations, contributing to
genome disorganization, expression changes, and chromosomal
instability. There is however little evidence that such effects
occur at a gross scale in human cancers. Rather, as a rule, L1
repression is only partly alleviated. Unfortunately, many
techniques commonly used to investigate genetic and epigenetic
alterations in cancer cells are not well suited to detect subtle
effects elicited by partial reactivation of retroelements like L1
which are present as abundant, but heterogeneous copies.
Therefore, effects of L1 sequences exerted on the local chromatin
structure, on the transcriptional regulation of individual genes,
and on chromosome fragility need to be more closely investigated
in normal and cancer cells. In normal somatic human cells, transcription of retrotransposon
sequences like L1 and illegitimate recombination involving them
are suppressed, restricting their activity to
developing germ cells and placental tissues [ 1– 3].
Suppression of retroelement activity prevents not only
retrotransposition, but also various disturbances of
transcription by retroelement promoters, interference by
retroelement enhancers, the activity of retroelement-encoded
enzymes, and illegitimate recombination between homologous
elements. Moreover, while L1 sequences have the potential to
create genomic instability, they probably exert certain
beneficial, “symbiotic” effects. For instance, silencing of
retrotransposons in somatic cells may help to organize the genome
into macro- and microdomains with differential transcriptional
activity. Failure to silence retroelements in cancer cells could
therefore permit adverse activities of retroelements as well as
perturb any beneficial effects. The present paper summarizes current knowledge about L1 (LINE-1)
retrotransposons in human cancer. For comparison, some
observations on human endogenous retroviruses (HERV) are included
[ 4]. Throughout the text, the emphasis will be on identifying
open questions, of which there are plenty, as should become
evident. Since L1 general biology is treated in detail in recent reviews
[ 1– 3] and other contributions in this issue, only a short
introduction will follow here. L1 sequences represent the major
class of LTR-less retrotransposons in humans and constitute about
18% of the human genome. While they are interspersed
throughout the genome, including euchromatic and heterochromatic
regions, they are particularly frequent in gene-poor regions that
correspond to chromosomal G-bands. Full-length elements are
6 kb in size and contain an internal promoter at the 5′-end
that generates a genomic transcript which also serves as an mRNA.
The RNA contains two open reading frames, ORF1 and ORF2. ORF1
encodes p40, an RNA-binding protein with cis preference for L1
RNA. ORF2 encodes the endonuclease and reverse transcriptase
required for retrotransposition. Only a fraction of L1 elements in
the human genome are intact. Most are truncated, usually at the
5′-end, and mutated, often at many sites. The up to 400, 000
elements that are still distinctly recognizable as L1 can be
categorized into several subclasses. Most and perhaps all elements
still capable of retrotransposition belong to a subclass named Ta.
Normally, transcriptional activity of L1 is restricted to
developing germ cells and to cells of the placenta. In somatic
cells, L1 transcription and retrotransposition is prevented by a
variety of control mechanisms, including methylation of L1 DNA and
specifically L1 promoters. In the germline, these mechanisms are
relaxed, and retrotransposition does occur occasionally. Potential dangers Dangers resulting from L1 reactivation in cancer cells comprise
the direct adverse effects of retrotransposition, enhanced
illegitimate recombination, and multiple ways of disturbance of
transcriptional activity and gene regulation. In the human genome,
fewer than 100 L1 elements are thought to be sufficiently intact
for retrotransposition [ 3]. However, while the danger of
retrotransposition is posed only by these intact L1s, other
adverse effects can be exerted by a larger number of elements. In
addition, reactivation may interfere with potential “symbiotic”
effects of L1 sequences such as their contribution to the global
and local organization of the genome and the provision of gene
regulatory sequences. Activities on the immune system can also be
envisioned. These would be expected to have ambiguous
consequences. It seems therefore imprecise to consider alterations
of L1 in cancer solely as “reactivation,” other effects may more
appropriately be characterized as “dysregulation.” Retrotransposition The mechanisms involved in L1 retrotransposition are now quite
well understood [ 1– 3]. The endonuclease encoded by L1 ORF2
induces single-strand breaks at AT-rich DNA target regions,
preferably at consensus TTTT/A sites. Following L1 ORF2
endonuclease action, the L1 RNA poly-A sequence pairs with
oligo-dT sequences in the target DNA, which serve as primers for
reverse transcription by the L1 ORF2 encoded enzyme. Reverse
transcription yields a branched DNA structure, which is presumably
resolved by cellular DNA repair systems. The retrotransposition
mechanism thus requires at least one recombination and creates two
DNA single-strand breaks close to each other, which can in effect
behave like a double-strand break. Therefore, attempted or
successful retrotranspositions carry a high risk of eliciting
chromosome breaks, deletions, translocations, and recombinations
[ 5]. Moreover, successful retrotransposition events are
likely to change the activity of genes at the insertion site.
Diverse outcomes of insertions are conceivable, including
increased or decreased transcriptional activity and the generation
of novel, variant transcripts (). On a
genome-wide scale, the effects of retrotranspositions might be
mitigated by the propensity of L1 elements to insert into AT-rich,
gene-poor regions of the genome, and especially into or close to
other elements [ 6]. However, even retrotranspositions outside
transcriptional units can have catastrophic effects on a cell by
inducing DNA strand breaks and initiating a breakage-fusion-bridge
cycles [ 7]. Effects on transcription Theoretically, a wide range of effects on the transcription of
host genes can be exerted by L1 regulatory elements and
transcriptional sequences that are located close to or within them
(). The L1 promoter is moderately strong
[ 8, 9] and the polyadenylation signal is relatively weak
permitting a substantial amount of read-through [ 10, 11].
Therefore, active L1 promoters located in sense orientation
5′ to a gene could override the normal transcriptional
controls of a gene to deregulate expression. Active L1 promoters
located in sense orientation within the transcriptional unit could
generate alternative 5′-truncated transcripts. Indeed,
many regulatory elements of human genes are derived from L1 or
HERV sequences [ 12]. Even some protein-coding sequences are
derived from retroelements. A prominent example is
syncytin1, a crucial protein required for the formation of syncytic
cells in the placenta which has evolved from an HERV env
protein [ 13]. The gene encoding syncytin1 is now consequently
named ERVWE1 for “endogenous retrovirus W envelope protein1.” As in this case,
regulatory sequences derived from L1 or HERV sequences are often
more active during germ cell or embryonic development than in
somatic cells. In cancer cells, decreased methylation and a more
open chromatin structure of such sequences could therefore allow
the reexpression of genes or transcripts that are normally
restricted to germ cells or the embryo, that is, oncofetal or
cancer-testis gene expression. Alternative transcripts may also be generated by the use of
polyadenylation sites of intragenic L1 sequences, especially if
these are 5′-truncated. As mentioned above, L1 polyadenylation
signals are weak. It is not known which mechanism ensures that
they are normally ignored in elements located within a
transcriptional unit. Consequently, it is difficult to estimate
how altered methylation and chromatin structure in cancer cells
would affect their recognition. Retroelements oriented in opposite direction inside a
transcriptional unit might interfere with transcription by
antisense effects, most prominently through formation of dsRNA.
This mechanism is implicated in the generation of heterochromatin
in some organisms [ 14, 15]. In mammalian cells, dsRNA ought to
induce general cellular antiviral defense mechanisms, for example,
by activating PKR, or leads to the production of siRNAs and
gene-specific downregulation. Interestingly, transcripts
containing Alu sequences in sense direction appear to be edited
and consequently destabilized in human cells [ 16– 18]. This
mechanism provides an obvious means of posttranscriptional gene
regulation. It is possible that a similar process acts on L1
sequences, but it is currently unknown to what extent intact or
partial L1 sequences in pre-mRNA are edited and whether such
sequences are employed for posttranscriptional gene regulation,
in normal or in cancer cells. A recent study [ 19] suggests
that L1 RNAs are not edited, at least not by the usual APOBEC3G
cytidine deaminase. A further possibility has been suggested by the recent discovery
of an antisense promoter near the 5′-end of intact L1
sequences [ 20]. When active, this promoter could exert
several effects on cellular genes, depending on its orientation.
If located in sense direction, antisense transcripts could lead to
downregulation; if located in antisense direction, it might lead
to overexpression of normal transcripts or the emergence of novel
transcripts. Accordingly, demethylation of L1 sequences in cancer
cells may not only activate their canonical sense, but also their
antisense promoters [ 21]. Effects of L1-encoded proteins Intact L1 elements contain two open reading frames. ORF1 encodes a
p40 RNA-binding protein supposed to act as a chaperone and
transport factor for L1 RNA. ORF2 encodes an endonuclease and a
reverse transcriptase. The properties of these enzymes have
meanwhile been studied quite well in vitro [ 22– 24], but
their impact on normal and cancer cells remains difficult to
estimate. One open question is how many L1 elements are actually
capable of expressing active proteins, especially, whether only
intact elements form the source. It is thought that less than
100 L1 sequences are capable of retrotransposition which all
belong to the Ta family [ 25]. They are the most likely source
of reverse transcriptase, endonuclease, and p40 protein in germ
cells and the embryo as well as in cancer cells. However, many
elements of other families are also intact, except for missense
and stop mutations. They could still give rise to one or the other
intact protein, as well as variant proteins. Alu
retrotransposition uses the enzymatic machinery provided by L1 and
is therefore dependent on expression of L1 proteins [ 26, 27].
Similarly, complementation of transposition in trans among L1
sequences is inefficient, but not impossible [ 28], that is,
full-length L1s with mutated protein-coding sequences might still
be capable of retrotransposition, if proteins are supplemented by
other elements. The proteins provided by L1s are also most likely
involved in the formation of pseudogenes. It is unknown, however,
whether their endogenous expression levels in cancer
cells are sufficient to support retrotransposition. Importantly, the potential danger of proteins encoded by L1s
depends critically on their ability to exert effects beyond aiding
retrotransposition in cis or in trans. In the context of cancer,
dangers posed by the endonuclease are most obvious. The
endonuclease introduces single-strand breaks “(nicks)” into DNA
with moderately stringent specificity [ 23]. Its activity is
further restricted by chromatin structure [ 22]. The ultimate
result of single-strand breaks introduced by the endonuclease in a
cell depends on several factors. A first factor is the cell cycle
phase. Nicks in S-phase are most problematic, because they can be
converted into double-strand breaks by the replication complex.
DNA repair competence and capacity
constitute a second factor that may differ between normal and
cancer cells. Thirdly, the presence of L1 RNA and other proteins
at the nicked site would be thought to influence the type and
efficiency of repair. The potential impact of L1 reverse transcriptase and RNA-binding
protein similarly depend on their specificity, actually in two
respects. First, to which extent are they specific for L1 (and
Alu) sequences? Second, are reverse transcription and
RNA-chaperoning their sole activities? Drug inhibitors of reverse
transcriptase and, more specifically, siRNA directed against L1 RT
decrease the proliferation of cancer cell lines [ 29]. Such
effects are difficult to explain by the known function of the
enzyme in mediating L1 and Alu retrotranspositions. Illegitimate recombination Successful and abortive retrotransposition can create chromosomal
instability and initiate illegitimate recombination by inducing
DNA strand breaks and by generating a branched DNA structure.
However, even in the absence of retrotransposition, the presence
of thousands of intact, rather long (6 kb), and relatively
homologous sequences in the genome plus the presence of
ten thousands of truncated and mutated sequences carries a
permanent risk of illegitimate recombination between elements
located at different sites. In the germline, recombination
between different L1 elements contributes to human evolution, but
also elicits inherited diseases. In somatic cells, recombination
ought to be restricted strictly to homologous recombination repair
of DNA double-strand breaks using homologous sequences from sister
chromatids or at most from the homologous chromosome. Any other
recombination event involves deletions, insertions, or
translocations. It is generally assumed that recombination between
L1 sequences in somatic cells is suppressed by dense DNA
methylation and tight packaging into chromatin. Decreased
methylation and relaxed chromatin structure of L1 sequences in
cancer cells might therefore facilitate illegitimate recombination
contributing to chromosomal instability. Disturbance of normal genome organization L1 sequences are thought to be
involved in the organization
of the human genome, their presence influencing short-range and
long-range chromatin structures. L1 sequences are overrepresented
in the late-replicating G-bands of human chromosomes [ 30]. It
is plausible that their presence is responsible for their more
heterochromatic character. L1s are also overrepresented on the
X-chromosome [ 31] where they may act as “way stations”
during X-chromosome inactivation [ 32, 33]. In a similar
fashion, methylated L1 sequences on other chromosomes which are
packaged into hypermethylated and deacetylated chromatin may
constitute the cores of localized facultative heterochromatic
regions. A fraction of centromeric heterochromatin also consists
of retrotransposons, mostly of L1s [ 34]. Intriguingly, some
L1 sequences are associated with nuclear matrix attachment regions
[ 35] and may contribute to the organization of chromatin
loops. L1 clusters located between genes may furthermore
contribute to the segmentation of the genome into transcriptional
units, helping to prevent interference by regulatory elements from
neighboring genes (). Such a “boundary”
function would explain why HOX clusters, which require long-range
interactions for their proper expression pattern, are largely
devoid of retroelement sequences [ 30]. Importantly, the
organization of the genome into subregions and loops pertains not
only to transcription, but also to replication and imposes
restrictions on the extent of DNA repair and recombination. Accordingly, alterations of DNA methylation and chromatin
structure at L1 sequences in cancer cells could have effects not
only on transcription, but also on DNA replication timing and on
the extents of recombination and DNA repair. Deregulation of gene
expression could not only be caused by activation of L1 elements,
but also through altered chromatin structure at inactive L1s
allowing transcriptional interference by neighboring enhancers or
silencers. Not only in this particular situation DNA replication patterns could be disturbed, with
normally late-replicating DNA shifting towards earlier periods
within S-phase. Barrier functions of repeat DNA in the genome
could be alleviated, allowing DNA processing during repair and
Holiday junctions formed during recombination to pass through
stretches of DNA that are less accessible in normal cells
[ 36]. Effects on cell stress and immune responses Endogenous retroelements have been implicated in the regulation of
cell stress responses, of the immune system, and in the
pathogenesis of several human autoimmune diseases. The strongest
data on regulation of human retroelements by cell stress
concern Alu sequences [ 37]. Likewise, the most convincing
data on regulation of the
immune system by retroelements and on the involvement in
autoimmune diseases implicates HERVs [ 4]. There are, however,
indications that L1 sequences too are induced during stress
responses [ 37], during cytotoxic chemotherapy [ 38], and
by UV exposure of skin cells [ 39]. Furthermore, L1 sequences
may act in a similar fashion as HERVs in at least one autoimmune
disease, rheumatoid arthritis [ 40]. In this disease, synovial
fibroblasts become aberrantly activated in a fashion that
resembles in many respects fibroblast activation in the stroma of
malignant tumors, with enhanced proliferation, migration, and
secretion of cytokines, chemokines, and proteases. The fibroblast
genomes at large and L1 promoters in particular were found to
become hypomethylated. Concurrently, full-length L1 RNA could be
detected [ 41]. Overexpression of the p40 ORF1 protein in this
disease has been suggested to activate stress-induced protein
kinases [ 42]. It is thought that L1 hypomethylation and
expression provide an amplification step in the pathogenesis of
the disease by enhancing immune responses [ 40]. The function of the activation of retroelements during cellular
stress responses is poorly understood. Conceivably, it forms part
of a signaling system that alerts the immune system to the
presence of infected or altered cells [ 43]. If that
proposition is true, hypomethylation and activation of L1
sequences in cancer cells are likely to influence the immune
response to malignant tumors. In support of this idea, some HERV
proteins have been found to behave as tumor antigens [ 44, 45],
but it is not known whether proteins encoded by L1 do so too. A
similarly open question is to which extent hypomethylated repeat
DNA liberated from tumor cells elicits danger signals in cells
regulating the immune response. Interestingly, L1 activation is
considered as a cause of increased plasma DNA levels in tumor
patients [ 46]. Observations Many of the effects that can be envisioned to be exerted by
activated L1 retrotransposons have indeed been observed in the
human germline and during fetal development [ 1– 3, 47, 48].
In cancer cells, mainly three phenomena point towards a
reactivation of retroelements. Retroelement DNA sequences become
hypomethylated, transcripts as well as protein products can be
detected, and L1 sequences are located at sites of breakage and
recombination. For L1 retrotransposons, the most convincing data
are available for hypomethylation. Data on L1 expression are
scarce, in contrast to several reports on the expression of HERV
gene products. L1 sequences have been found at or near deletion
ends and translocation breakpoints, but the precise frequency and
the mechanisms involved remain to be determined. Intriguingly,
actual retrotransposition events are exceptional. Altered methylation In a large number of human cancers, decreased methylation of L1
sequences has been documented ( Table 1). This decrease
occurs in the context of general alterations in DNA methylation
patterns that accompany carcinogenesis in many human tissues.
These are regarded as part of an important epigenetic mechanism
driving cancer development and progression [ 63]. Alterations
of methylation in cancer cells comprise “hypermethylation” which
occurs focally and in a largely specific fashion, typically at
CpG islands surrounding the transcriptional start regions of
individual genes. Somewhat paradoxically, in many, but not all
cancers, increased methylation at specific sites is found
alongside a decrease in methylation levels of the overall genome.
The decrease in methylation appears to be relatively unspecific
and is therefore commonly designated as “genome-wide” or
“global” hypomethylation [ 64, 65]. In normal somatic cells,
the bulk of methylcytosine is found in repetitive sequences such
as L1, HERVs, and Alus, but also at CpG-rich satellites such as
SAT2 and SAT3. The overall decrease in methylation found in cancer
cells therefore reflects a largely parallel decrease in the
methylation of retrotransposon sequences [ 61]. As a rule,
however, L1 and HERV sequences seem to be more strongly affected
than Alus [ 65]. | Table 1Hypomethylation and expression of L1 in human cancers. |
Hypomethylation of L1 sequences has traditionally been
investigated by Southern blot analysis following digestion of DNA
with methylation-sensitive restriction enzymes [ 53].
Recently, methods employing PCR following bisulfite treatment of
DNA have been developed for this purpose [ 52, 66]. These
techniques are promising, since they can also be applied to small
amounts of suboptimal quality DNA. However, because of the
heterogeneity of L1 sequences, the extent of their demethylation
is difficult to estimate precisely, especially by PCR-based
methods. Southern blot analyses suggest that in cancer cell lines
up to 70%–80% of CpG sites in L1 sequences become
demethylated. Decreases in L1 methylation appear to parallel those
in HERVs. Accordingly, individual HERV proviruses are essentially
unmethylated in cancer cell lines with strong hypomethylation
[ 53]. Nevertheless, L1 hypomethylation is anything but
uniform in different cancers, in two respects. First, different
extents of hypomethylation are found in cancers of the same type.
These differences are also observed in cancer cell lines and are
therefore not explained by differences in the proportion of tumor
cells in tissue samples. Second, L1 hypomethylation appears to
develop at different stages in the development of different
cancers. For instance, it is found at early stages of colon and
bladder cancers [ 53, 60], but only in higher-stage prostate
carcinomas [ 55, 56] while primary renal carcinomas lack
significant LINE-1 hypomethylation [ 52, 53]. Germ cell cancers
are a special case since they have generally hypomethylated
genomes, presumably due to their origin from cells with lower
methylation levels [ 67, 68]. Accordingly, L1 [ 51, 69] and
HERV [ 70] sequences are strongly hypomethylated in testicular
cancers. Finally, note that very little is known on
the methylation of individual L1 sequences [ 71], and
accordingly, whether their hypomethylation in cancers is uniform
[ 72]. Although genome-wide hypomethylation in human cancers has been
known for more than twenty years, the mechanisms eliciting this
alteration are still unknown. Hypothetical mechanisms include
insufficient levels of methyl group donors, ultimately of
S-adenosylmethionine, inadequate expression or regulation of DNA
methyltransferases, reexpression of DNA demethylases, and altered
expression of chromatin regulators directing DNA
methyltransferases [ 64, 65]. The last mechanism is particularly interesting in the present
context. Retroelements constitute approximately 45% of the
human genome [ 30] and contain an at least proportionate
amount of methylcytosine. Moreover, they appear to be
preferentially recognized by the DNA methylation machinery
and—at least in some circumstances—appear to act as “centers of
methylation” from which methylation spreads into adjacent
sequences [ 73]. Therefore, genome-wide hypomethylation could
theoretically arise as a consequence of a defect in the
recognition of retroelements as methylation targets. Unfortunately, it is still not known how retroelements are
distinguished for silencing in mammalian genomes. The L1 promoter
is as active in somatic as in embryonic cells [ 9]. Therefore,
L1 silencing in somatic cells cannot be simply a consequence of
transcriptional inactivity. Instead, silencing must have been
actively established during fetal development and is faithfully
maintained through cell proliferation and differentiation in
normal somatic cells. DNA methylation of retroelements is
established first during germ cell development and then again
during gastrulation, when the genome at large becomes
de novo methylated, except for sequences that are actively
protected, such as CpG islands and active imprinted genes
[ 74]. De novo methylation in the mouse embryo requires DNA
methyltransferases, specifically Dnmt3A and Dnmt3B as well as
Dnmt1 for maintenance of the established methylation [ 74, 75].
In male germ cells, Dnmt3L is required for proper L1 methylation
[ 76]. It is not entirely clear whether methylation of L1
during development requires specific chromatin regulators
directing the methyltransferases. One candidate is SMARCA6, as its
mouse orthologue Lsh has been found to be required for proper
methylation of L1 sequences. Inactivation of Lsh in mice causes L1
hypomethylation, but only limited disturbances of the methylation
of single-copy genes [ 77]. In comparison, inactivation of
another chromatin protein ATRX causes hypomethylation of rDNA, but
leaves L1 methylation intact [ 78]. This suggests that the
specificity of DNA methylation may be regulated by specific
“chromatin regulator” proteins. A variety of chromatin regulator proteins have been reported to be
aberrantly expressed or even mutated in human cancers
[ 65, 79– 81]. However, many of these changes are rare or are
specific to particular cancers. It is therefore difficult to
envision a change in a single “master regulator” of L1
methylation as the cause of the widely distributed hypomethylation
of these sequences. More likely, L1 hypomethylation could be
associated with the general reorganization of chromatin structure
in aneuploid cancer cells that disturbs the compartmentation of
the genome [ 65, 79, 80]. Genome-wide alterations in histone
modification have recently been described in cancer cells
[ 82, 83]. Given the high proportion of L1 sequences in the
human genome, these are likely to affect these retrotransposons
and to interact with their methylation.
Note that the relation between DNA methylation and histone
modifications at L1 sequences is far from being understood [ 84]. L1 expression in cancers The mechanisms underlying hypomethylation changes in human cancers are not
understood, but even the description of these changes is
fragmentary. For instance, methylation of HERVs has been studied
in only a few cancers. Available data suggest that they are
affected by genome-wide hypomethylation in parallel to LINE-1
sequences ( Table 1). In selected cancers, endogenous
retroviral sequences may be almost completely unmethylated.
Expressed sequences derived from HERVs are found in germ cell
cancers and antibodies directed against HERV-encoded proteins are
found in the blood of patients [ 70]. In cancers of somatic
cell origin, bona fide transcripts for envelope and auxiliary
proteins have been reported, especially in breast cancer
[ 44, 85], and recently in melanoma [ 86]. Some results
suggest that HERV expression occurs in a wider range of cancers
and even normal tissues [ 87, 88]. These data need further
verification to exclude artifacts from genomic DNA and unspliced
transcripts. Moreover, the somewhat surprising findings that
different transcripts from different subfamilies may be expressed
in a cancer-type-specific fashion call for a closer analysis of
the mechanisms involved. There are no sufficiently systematic studies of L1 expression in
human cancers. The available data suggest that expression of
full-length L1 sequences is by far the strongest in
teratocarcinomas, while weaker expression is observed in a wider
range of carcinomas exhibiting hypomethylation [ 53]. This
expression pattern therefore resembles that of HERVs. Since HERVs
also give rise to spliced transcripts, RNA analyses can provide a
first indication of which protein products are expressed. For L1,
this question needs to be addressed using antibodies. So far, no
definite data have been published on the expression of the
proteins encoded by the retrotransposons in human cancer. Their
presence in germ cell cancers and teratocarcinoma cell lines,
however, is very likely [ 89]. Involvement of L1 in chromosome breakage and
recombination Whereas retrotransposition events take place quite regularly in
the germline, at an estimated rate of 1 event per 100 births
[ 3, 4], very few have been reported in cancer cells
[ 90, 91]. Similarly, although L1 sequences have been shown to
become incorporated at sites of double-strand break DNA repair in
model experiments [ 92], according sequence changes have only
exceptionally been observed in human cancers [ 93]. In spite
of the caveats discussed below, it is therefore probably safe to
conclude that actual retrotransposition events are rare in human
cancers and do not regularly contribute to genomic instability. The evidence is better for indirect mechanisms by which
retrotransposons could promote chromosomal instability in human
cancer. L1 hypomethylation and chromosomal instability correlate
well with each other in several cancer types [ 55, 58, 94]. A
similar relationship has been observed between the hypomethylation
of tandem satellite sequences and alterations of the chromosomes
that carry them as large juxtacentromeric region [ 95– 97].
In this case, hypomethylation of the satellite sequences is
thought to cause decondensation of pericentromeric chromatin and
an increased propensity for chromosomal breaks and rearrangements
in this region. In a similar fashion, hypomethylation of
retroelement sequences dispersed in the genome could facilitate
illegitimate recombination. In favor of this idea, L1 sequences are
enriched at the ends of 3p14.1 and 9p21 deletions in carcinomas
[ 36, 98, 99] and homozygous deletions arise preferentially in
chromosomal regions with high LINE content [ 100]. It has also
been suggested that L1 and HERV sequences are involved in the
formation of double-minute circular chromosomes in cancer cells
[ 101, 102]. The most straightforward hypothesis accounting for these findings
is that decreased methylation and presumably more open chromatin
structure at L1 sequences in cancer cells favors the illegitimate
recombination between elements at different genomic locations, for
example, during homologous recombination repair of DNA strand
breaks. However, closer analyses of the deletion ends in solid
tumors indicate that this hypothesis is probably incorrect. While
deletion ends are indeed often located in or near L1 sequences and
particularly L1 clusters, the breakpoints invariably show
hallmarks of DNA double-strand break repair by nonhomologous
end-joining (NHEJ). Typically, one end of the deletion is located
in or close to an L1 sequence, while the other end is provided by
an unrelated single copy or repeat sequence [ 36, 99, 103]. Such
structures also appeared as occasional end products of repair of
DNA double-strand breaks induced by a restriction endonuclease at
a specific chromosomal site [ 104]. A plausible
explanation for this structure is that processing
by the NHE1 protein complex
damaged DNA
ends is slowed down at L1 sequences by denser chromatin, favoring
reannealing and ligation there [ 36]. If this explanation is
correct, retroelement hypomethylation in cancer could
paradoxically diminish the tendency of breakpoints to be located
at L1 sequences. It would instead tend to increase the size of
deleted and recombinated sequences, because DNA processing and
Holiday junctions arising during recombination repair could move
further through more open chromatin. Presently, either hypothesis remains speculative for several
reasons. First of all, far too few chromosomal breakpoints have
been investigated, especially in carcinomas. Secondly, it has not
been established for any chromosomal alteration whether
hypomethylation of repeat sequences at the affected site preceded
it. Thirdly, L1 repeats are not randomly distributed in the
genome. They might be associated with local structures that are
particularly prone to breakage, such as fragile sites or the
anchorage sites of chromatin loops. Perspectives Consequences of L1 activity in the human germline are well
documented. Retrotranspositions in the germline take place at a
significant rate of approximately 1 event per 100 births [ 3].
In addition, a substantial number of recombination events
involving L1 elements have been detected, typically because they
elicited translocations or rearrangements causing inherited
diseases [ 2, 47, 48]. Specifically, L1 retrotransposition and
illegitimate recombination in the germline are causes of inherited
and congenital cancers. For instance, a germline
deletion in the MLH1 gene carries hallmarks of
recombination initiated by a failed L1 retrotransposition event
[ 105]. In contrast, in spite of considerable evidence hinting at a
reactivation of L1 retrotransposons in a variety of human cancers,
there is limiting evidence for major consequences of this process.
This may be due to two very different reasons. One is technical:
even typical effects expected from L1 reactivation are difficult
to detect by the techniques commonly used to investigate genetic
alterations in human cancers. The other is biological:
reactivation may be partial and the mechanisms ensuring silencing
of L1 DNA sequences and limiting the effects of transcribed
sequences may remain functional to some degree. Perhaps, limited
reactivation of L1 sequences may exert effects more through the
loss of symbiotic functions than through direct adverse effects on
genomic stability. This possibility is even more difficult to
ascertain. In general, investigations of genetic and epigenetic changes in
human cancers avoid dealing with repeat sequences and focus on
single-copy protein-coding genes. Mutation analysis of genes is
typically restricted to coding sequences and employs PCR
techniques to analyze individual exons or mRNA. Insertions or
recombinations caused by L1 or other retroelements would
often not be detected by this approach, unless they occur within
exons. Therefore, it might not be coincidental that reports
describing oncogene activation and tumor suppressor inactivation
by L1 insertion date from a period when Southern blot analysis was
more en vogue. Similarly, recombination and deletions in cancers are well
documented at the level accessible by cytogenetic techniques, but
are not well investigated at the molecular level, with the
important exception of translocations in hematological cancers. In
these, retroelements have indeed been found at many translocation
sites, although their role in the generation of the translocations
is not clear. In contrast, very few studies have addressed the
precise structure of chromosomal breakpoints in solid tumors. A
recent genome-wide study of 505 cancer cell lines yielded a strong
association between LINE content and the presence of homozygous
deletions, but no breakpoints were characterized in detail
[ 100]. Detailed analyses of deletion endpoints at the FRA3B
fragile site [ 98] and around CDKN2A at 9p21 [ 99]
revealed a preponderance of L1 sequences at or close to the
deletion endpoints. Such analyses remain tedious even with the
finished human genome sequence having become available. Therefore,
we know little on the structure of amplicons, another category of
unstable sequence in cancer cells, and next to nothing on the
sites of illegitimate recombination in cancer cells. L1 sequences
have been detected in double minutes, an important intermediate in
one amplification mechanism, and have been proposed, but not proven to
be involved in their formation [ 101]. By a comparison of loss
of heterozygosity analysis and cytogenetic techniques of
chromosome 8p in bladder cancer cell lines, recombination events
were recently shown to be much more frequent and were shown to take place
across much smaller regions than hitherto assumed [ 106].
However, it is not known and difficult to determine what initiated
the recombination events and which sequences precisely were
involved. In summary, therefore, whereas it seems unlikely that
retrotransposition is common in human cancer cells, the role of
L1s in recombination and chromosome breakage is probably
underestimated due to a lack of studies with appropriate
methodology. A similar argument can be made for epigenetic effects of L1
sequences in cancer. In genome-wide screens for altered
methylation in cancer, repeat sequences are often and
understandably considered a nuisance and typically removed by
prehybridization. Overall changes in L1 methylation are therefore
well documented, but data on the behavior of individual sequences
is lacking. Bisulfite sequencing is restricted to a few hundred bp
per PCR and is prone to artifacts from template switching and
target priming when applied to repeat sequences. An elegant
solution may be hairpin PCR. This method has revealed that in
fetal fibroblasts, the promoters of most full-length L1 sequences
are densely and symmetrically methylated, while selected elements
are unmethylated [ 71]. The obvious question is which elements
are these. Accordingly, it is not clear whether the number of
completely unmethylated elements increases in cancer cells or
whether the decrease in methylation is distributed across all L1
sequences. These questions extend of course to the issue of
chromatin structure at L1 elements. The contribution of L1s to altered gene expression in cancer is
still more difficult to ascertain. There are many unexplained
instances of altered gene expression in cancers. Perhaps most
striking are reports on frequent downregulation of genes (usually
tumor suppressor candidates) without detectable genetic
alterations in their vicinity and altered DNA methylation in
regulatory sequences. Recently, increased expression of miRNA has
been introduced as a potential cause of such enigmatic
observations [ 107]. In the light of potential effects of L1
sequences, perhaps effects exerted by L1 elements in or near
affected genes should also be considered. This suggestion likewise
applies the mechanisms generating aberrant transcripts in cancer
cells [ 21]. Again, this is a highly difficult issue,
especially if genes that are investigated are alternatively
spliced even in normal cells. The argument can be broadened
further to encompass the potential boundary function of repeat
elements. Its disturbance by altered chromatin structure in cancer
cells may result in more or less subtle up- and downregulation.
More than two decades of intense work have been spent on a small
number of selected loci to understand the mechanisms of action of
long-range regulatory elements and boundary elements at all. It is
understandable that very little is known on how they are altered
in cancer cells. Still, it may be advisable to consider such
effects and others mentioned in this paragraph when encountering
instances of altered gene regulation in cancer that cannot be
straightforwardly explained by mutations or altered DNA
methylation of gene regulatory sequences. The difficulties in determining the impact of L1 sequences in
cancer cells resulting from methodological limitations are
compounded by biological factors. Several layers of mechanisms
control L1 expression and activity. DNA methylation inactivates
the L1 promoter [ 9]. It is likely that this restriction of
transcriptional activity is aided by an inactive chromatin
structure [ 108], although this cannot be considered proven
[ 86]. A second set of L1 controls appears to act at the RNA
level, perhaps exerted by cytidine deaminases, leading to RNA
instability [ 11]. Alu-containing RNAs are subject to editing
[ 16– 18], but the evidence for L1 RNA is scanty. A third
level of control is enacted at the retrotransposition step. Active
TP53 prevents retrotransposition [ 109], but this is very
likely not the only barrier at this step. Last and perhaps not
least, there is some evidence that retroelement activation might
attract immune responses. As discussed elsewhere in detail
[ 43], such responses are better documented for HERVs, but
they might additionally or concurrently select against cells with
strongly activated L1 retrotransposons outside immunoprivileged
organs. There is good evidence for three of these protective mechanisms to
be impeded in cancer cells: DNA methylation is decreased, TP53 and
checkpoints are often defective, and immune responses to
advanced cancers are muted. We know very little about another
mechanism, control of retroelements at the RNA level. In summary,
therefore, the regulation of L1 genomic structure, expression, and
retrotransposition is clearly perturbed in many human cancers, but
inactivation of all tiers of control may be rare. Some cancer
types exhibit few changes, for example, renal cell carcinoma,
where even L1 DNA methylation appears to be maintained, while germ
cell cancers appear to represent the other end of the spectrum
[ 49, 50, 52, 53]. Even in these, however, retrotransposition
events appear to be rare and the evidence for major contributions
of retrotransposon activity to the cancer phenotype is limited.
Presumably, at least one of the multiple safeguards against
retrotransposition holds up. A likely candidate is TP53
[ 109], since germ cell tumors are among the few cancer types
in which mutations of this tumor suppressor are rare [ 68]. Activation of L1 retrotransposons in cancer cells is expected to
exert a variety of effects on the tumor phenotype, if it occurs.
Of course, this statement hinges on the “if,” and our present
knowledge does not allow firm conclusions. Considering that L1
retrotransposons make up almost a fifth of our genome, there are
astonishingly large gaps in our knowledge on their general
biology, and consequentially in our knowledge on their behavior in
cancer. As argued above, there is an obvious need for more
systematic investigations of DNA methylation and chromatin
structure of L1 DNA, of the expression of full-length transcripts
and L1-encoded proteins on one hand and for exemplary studies of
individual elements and their influence on adjacent genes in
cancer cells on the other hand. At this stage, it is probably safe
to conclude that L1 retrotransposons do become reactivated to
various degrees in different cancers, but that some of the many
safeguards that prevent retrotransposition and their adverse
effects in somatic cells hold up in most cancers. Perhaps, even
cancer cells cannot survive with fully active retrotransposons. It
follows that more subtle effects of L1 dysregulation in cancer
cells, which may include adverse actions as well as loss of
symbiotic functions, should be a focus of investigation. I am grateful to Dr. Andrea R. Florl for critical reading of the
manuscript and to Sandy Fritzsche for help in compiling the
reference list. Work in our lab is supported by the Deutsche
Forschungsgemeinschaft (LI 1038/3-1) and the Deutsche Krebshilfe
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